Antiviral agent comprising CD4 and H2 histone

ABSTRACT

The invention provides an agent against HIV and/or related viruses, comprising CD4 or a CD4-like substance and an H2 histone or an H2 histone-like protein. The content of the CD4 or CD4-like substance is preferably less than the antivirally effective dose of that substance alone. The invention also provides an H2 histone or an H2-like protein, for use in a method of medical treatment, in particular against HIV and/or related viruses, and also for use in the manufacture of a pharmaceutical composition against HIV and/or related viruses.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to anti-viral agents, especially agents activeagainst HIV-1 and related viruses.

2. Description of the Related Art

It is known that an initial stage of at least one mode of the infectionof humans by RNA and DNA viruses involves attachment of the virus to themembrane of a human cell. In the case of HIV-1 and HIV-2, binding isbetween a cell surface glycoprotein, known as CD4, and a viral envelopeprotein known as gp120. Thus, CD4 acts as a cell surface receptor forthe virus.

After attachment of the virus to the cell membrane the replication cycleof the virus involves a series of steps, one of which appears to befusion of the viral envelope with the cell membrane. It might beexpected that fusion depends solely on binding of gp120 to CD4. However,it is known that if the gene for human CD4 is transfected into CD4negative HeLa.cells, these cells are then rendered susceptible toproductive infection by HIV-1, whereas transfection of the same geneinto mouse L-cells causes these cells to bind the virus but does notallow internalisation to occur. Also, experiments with VSV(HIV)pseudotypes have shown that HIV-1 binds to mouse L-cells transfectedwith human CD4 but is not internalised. Furthermore, it has beenreported that CD4 negative cells from a wide range of human tissues canbe infected by HIV-1; such infection is not blocked by preincubation ofthe cells with an anti-CD4 MAb (Leu 3a) or by incubation of the viruswith sCD4. These observations cause us to postulate that fusion of thevirus envelope with that of a CD4 positive cell requires the involvementof at least one other cell surface receptor, a "fusion receptor".

SUMMARY OF THE INVENTION

The present invention is based on our discovery that at least one suchfusion receptor appears to be a cell surface protein having N-terminalhomology to an H2 histone.

The invention provides an agent against human immunodeficiency virus(HIV) and/or related viruses comprising CD4 or a CD4-like substance (forexample, CD4-IgFc immunoadhesins, CD4 V1-V2 domains, and CD4-derivedpeptides) and an H2 histone or H2 histone-like protein. The content ofCD4 or CD4-like substance may be less than (for example, less thanone-half of) the anti-virally effective dose of the CD4 or CD4-likesubstance alone.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In therapeutic use, the anti-viral agent of the invention is normallyadministered parenterally, for example intravenously. However,administration via the peritoneal cavity may be the most effective routein that it results in entry of at least some of the anti-HIV agentdirectly into the lymphatic system, where viral replication may beextensive.

The invention also provides the use of the agent described above againstHIV and/or related viruses, the agent preferably being administeredintraperitoneally, especially in a solution containing an osmotic agentwhich is a glucose polymer mixture, derived from the hydrolysis ofstarch, which includes at least 50% of glucose polymers of a degree ofpolymerisation greater than 12, as described in our British patentspecification number 2154469A.

Further, the invention provides a pharmaceutical composition containingthe agent described above together with an inert carrier or diluent; italso provides the agent of the invention for use in the manufacture of apharmaceutical composition against HIV and/or related viruses.

The invention additionally provides a method of treatment of a human oranimal subject carrying the HIV virus and/or a related virus, comprisingadministering to the subject a pharmaceutically effective amount of theagent of the invention. The CD4 or CD4-like substance and the H2 histoneor H2 histone-like protein may be administered to a subject together orone after the other, in any order, although preferably with the CD4 orCD4-like material being administered before the H2 histone or H2histone-like protein.

The invention also provides an H2 histone or an H2 histone-like protein,for use in a method of medical treatment of a human or animal body, inparticular for use in a method of treatment of a human or animal subjectcarrying the HIV and/or a related virus; and an H2 histone or an H2histone-like protein, for use in the manufacture of a pharmaceuticalcomposition against HIV and/or related viruses.

It is known that some sulphated polysaccharides, such as dextransulphate, heparin, heparin fragments, pentosan polysulphate andfucoidan, block infection by HIV-1 in vitro of human CD4+ and CD4-cells. It seemed possible that in the case of CD4+ cells this anti-viralactivity involved interference with the attachment of the virus to theCD4 receptor on the host cell. However, it is now clear that mostnaturally occurring sulphated polysaccharides bind to sites which areseparate from and/or additional to CD4. The manner in which sulphatedpolysaccharides act is therefore still uncertain and it is possible thatthey do not all have the same mode of action.

We have hypothesised that at least some of the anti-viral activity ofsulphated polysaccharides in blocking HIV-1 infection is by acting on a"fusion receptor", a cell surface component which is present on all ormost human cells and is critical for the fusion process. Some sulphatedpolysaccharides may also attach themselves to viral gp120, but anyantiviral activity resulting from this interaction is probably of lessimportance than their activity in blocking the fusion receptor.

To investigate this hypothesis, we carried out a number of experimentsusing dextrin sulphate as the anti-HIV agent.

Dextrin is a mixture of glucose polymers, in which the glucan units havepredominantly alpha-1,4 glucosidic bonds. The dextrin sulphate used inour experiments was a 2-sulphated dextrin sulphate with a weight averagemolecular weight of about 27,000. It had a sulphur content of 12% byweight, equivalent to one sulphate group per glucose residue.

In tissue culture experiments we have found that dextrin sulphate blocksinfection of CD4+ cell lines (M8166, HPB ALL) by a variety of HIV-1laboratory isolates (IIIb, RF, MN) at an inhibitory concentration of6-12 ug/ml (230-460 nM). We have also shown that dextrin sulphate blocksinfection of several CD4- cells; TE671 HH24 (rhabdomyosarcoma), U251 SPHH51, U138 HH 54 (glia) and HT29/7 (colorectal carcinoma). Infection isnot prevented by preincubating the cells with Leu 3a or the virus withsCD4.

These tissue culture experiments appeared to support our hypothesis thatdextrin sulphate blocked HIV-1 infection by acting on a fusion receptor.We then carried out receptor-ligand binding studies, and found noevidence to suggest that dextrin sulphate binds to CD4. Preincubation ofCD4+ cells with Leu 3a does not alter the binding constants, andtransfection of CD4 into HeLa cells reduces rather than increasesdextrin sulphate binding. We have also found that pretreatment of HPBALL cells with trypsin abolishes dextrin sulphate binding. In contrast,pretreatment with neuraminidase does not affect binding, suggesting aprotein binding site. These results suggest that the same dextrinsulphate binding site is present on human CD4+ and CD4- human cells.

We have carried out ligand-blot experiments with a view to identifyingthe nature of the dextrin sulphate binding site. HPB ALL cell membraneswere prepared and the proteins separated using 12.5% SDS-PAGE gels.After blotting to nitrocellulose filters, they were probed withtritiated dextrin sulphate. Binding of the labelled dextrin sulphate tobands which correspond to proteins of molecular weights of 29 kDa and 17kDa was observed; binding of the labelled dextrin sulphate to these twobands could be displaced with an excess of cold dextrin sulphate. Mostof the displaceable binding was to the 17 kDa site.

It appears that one or other of the proteins to which dextrin sulphatebinds may constitute the fusion receptor or part of the fusion receptor.Determination of the N-terminal sequence of two of the proteins ofinterest, the 17 kDa proteins, revealed that one of them (withN-terminal sequence PEPAKSAPAPKKGXKKXVTKA) showed homology with histoneH2B and the other (with N-terminal sequence ARTKQTARKSTGGKAPRKQLAT) withhistone H3.1. There has been no previous evidence to suggest thathistones were in any way involved in the HIV infection cycle. Histonesare a group of strongly basic proteins.

We now believe that a histone or a histone-like protein may constitutethe fusion receptor postulated above. It is known that as nucleoproteinsH2A, H2B and H3.1 can bind DNA and RNA. We therefore believe that one orall of these histones or histone-like proteins on the cell surfaceinteracts with viral RNA, and that this leads to entry of the virus intothe cell. H2A and H2B are also known to bind heparin, a sulphatedpolysaccharide, so it is reasonable to assume that these histones willbind some other sulphated polysaccharides.

HIV infection of CD4 positive cells appears to be initiated by thebinding of the viral envelope protein gp120 to the cell surface proteinCD4, following which there is a temperature-dependent conformationalchange which exposes the hydrophobic, N-terminus of gp41 which isbelieved to fuse with the cell membrane. The mechanisms underlyingfusion remain to be defined. Morphological studies, however, suggestthat the process of fusion involves spontaneous disintegration of thecore and the release of viral RNA into the cytoplasm.

Our results suggest a mechanism of viral entry in which cell surfacehistones interact with a component of HIV-1 following the conformationalchange induced by binding to CD4. We have not established whether cellsurface histones interact with a core or an envelope component of HIV-1.However, the known anti-HIV activity of guanosine oligonucleotides andtheir ability to bind in a specific manner leads us to suggest thatguanosine-rich regions within viral RNA may be able to interact withcell surface histones. The histone-RNA complex formed could then undergomicropinocytosis and internalisation.

We think it possible that dextrin sulphate and guanosineoligonucleotides block HIV infection by binding to cell surface histonesand preventing their interaction with gp41.

The mechanism postulated above implies that the initial interaction ofgp120 with CD4 results in the uncoating of the virus particle. Other, asyet undefined, cell surface receptors (e.g. proteinases) may be involvedin this process.

Our experimental work has shown that neither calf thymus histones (H1,H2A, H2B, H3 or H4) nor other basic proteins (i.e. lysozyme andalpha-bungarotoxin) have any intrinsic anti-HIV-1 activity. However,when C8166 cells preincubated with histone H2A or H2B were added toHIV-1 (IIIB) which had first been incubated with sub-inhibitoryconcentrations of soluble CD4, the cells did not become infected. Theamount of soluble CD4 required to inhibit infection was progressivelyreduced with increasing concentrations of histone H2A or H2B. Incontrast, a similar antiviral effect was not seen in combinations ofeach of the histones H1, H3, H4, lysozyme and alpha-bungarotoxin withsoluble CD4.

Example 1, below, describes the results of experiments to examine theanti-viral activity of histones H2A and H2B, alone and together withsoluble CD4.

EXAMPLE 1

Calf thymus histones were used for these experiments because they areidentical or highly homologous with human histones. C8166 cells wereincubated with histone H2A or H2B (Boehringer Mannheim, Germany) for onehour at 37° C. At the same time, HIV-1 (IIIb; 10².5 culture infectiousdoses) was incubated with sCD4 (baculovirus) for 1 hour at 37° C. Thesymbol (+) represents no reduction in the number of syncytia counted inC1866 cells as compared with controls. The symbol (-) represents a 90%reduction in the number of syncytia as compared with controls. Tables Iand II summarise the results from three separate experiments.

In the absence of CD4, the histones are inactive even at 62.5 ug/ml. Inthe absence of the histone, CD4 inhibited HIV-1 infection at 0.5 ug/ml;the activity of CD4 was enhanced by the presence of histone, suchenhancement being increased with increasing concentration of thehistone.

                  TABLE I                                                         ______________________________________                                                   Soluble CD4 (ug/ml)                                                Histone H2A (ug/ml)                                                                        0.5   0.375    0.25 0.188  0.125                                                                              0                                ______________________________________                                        62.5         -     -        -    -      -    +                                46.9         -     -        -    -      +    +                                31.25        -     -        -    -      +    +                                23.4         -     -        -    +      +    +                                15.6         -     -        -    +      +    +                                11.7         -     -        -    +      +    +                                7.8          -     -        +    +      +    +                                5.9          -     -        +    +      +    +                                3.9          -     -        +    +      +    +                                2.9          -     +        +    +      +    +                                1.95         -     +        +    +      +    +                                0.0          -     +        +    +      +    +                                ______________________________________                                    

                  TABLE II                                                        ______________________________________                                                   Soluble CD4 (ug/ml)                                                Histone H2B (ug/ml)                                                                        0.5   0.375    0.25 0.188  0.125                                                                              0                                ______________________________________                                        62.5         -     -        -    -      -    +                                46.9         -     -        -    -      -    +                                31.25        -     -        -    -      +    +                                23.4         -     -        -    +      +    +                                15.6         -     -        -    +      +    +                                11.7         -     -        -    +      +    +                                7.8          -     -        +    +      +    +                                5.9          -     -        +    +      +    +                                3.9          -     -        +    +      +    +                                2.9          -     +        +    +      +    +                                1.95         -     +        +    +      +    +                                0.0          -     +        +    +      +    +                                ______________________________________                                    

From these tests it appears that the histones themselves have noanti-HIV activity at relatively high concentrations. However, they boostthe activity of CD4, so that anti-HIV compositions which rely for theiractivity on the presence of CD4 can be formulated with a lower CD4content for the same activity if they contain one of these histones inaddition to CD4. It is believed that the srCD4 causes a conformationalchange in the Viral envelope of the virus from the capsid, and infectionis then blocked by H2A and/or H2B. Enhancement of the anti-HIV activityof CD4 is not seen with other histones or with other basic proteins(lysozyme and alpha bungarotoxin).

We claim:
 1. A composition comprising CD4 or a CD4-like substance and anH2 histone or an H2 histone-like protein and a pharmaceuticallyacceptable carrier.
 2. The composition of claim 1 wherein the histone ishistone H2B.
 3. The composition of claim 1 wherein the histone ishistone H2A.